The bHLH transcription factor Ascl1 (previously Mash1) is essential for neuronal differentiation and sub- type specification of multiple neuronal cell-types throughout the brain, spinal cord, and autonomic nervous system, as well as neuroendocrine cels and cels in sensory systems such as the retina and olfactory epithelia. Ascl1 function is balanced with that of Notch signaling to control progenitor proliferation and differentiation. In this respect, it is not surprising that Ascl1 expression is aberrantly present n neural and neuroendocrine tumors, and has also been identified as a key factor in directly reprogramming fibroblasts to neurons. With these important functions attributed to Ascl1, it is of fundamental importance to understand how Ascl1 functions as a transcription factor in these processes. There has been a tremendous advance in defining the importance of genomic landscape and epigenetics in controlling lineage specific gene expression programs. However, in most cases, connecting the chromatin modifications with site-specific DNA binding factors like Ascl1 is lacking. At the end of the previous funding period, we succeeded in generating a map of Ascl1 bound sites across the genome in vivo during neural tube development using chromatin immunoprecipitation followed by sequencing (ChIP-seq). These data revealed the identity of multiple factors that may play important roles as collaborating factors modulating Ascl1 activity. In the next funding period, we propose to test models for how Ascl1 interfaces with the genome and with collaborating factors to regulate lineage specific gene expression required to understand and manipulate neural lineage specific gene programs. Project goals are to 1) identify transcription factors collaborating with Ascl1 in neural development, 2) distinguish between models of chromatin accessibility and sequence specific mechanisms for how Ascl1 functions to activate a neural gene expression program, and 3) test the hypothesis that a novel Ascl1-interacting factor connects site-specific DNA binding factors like Ascl1 with higher order chromatin modifications to regulate downstream gene expression programs. Understanding how Ascl1 functions as a transcription factor and identifying co-factors that modulate its activity and the gene programs it regulates will provide key entrance points for improving the efficiency of generating specific types of neurons in vitro and in vivo, and for targeting tumor generating cells PUBLIC HEALTH RELEVANCE: Alterations in the balance of neural progenitor maintenance with differentiation, and the balance of neuronal types generated are thought to underlie diverse neurological disorders from autism to cancers of neural origin. Ascl1 is an essential regulator of this balance in multiple regions of the nervous system in the embryo and the adult, and is a key factor in reprogramming cells to neurons. How Ascl1 functions with other regulatory factors to regulate the neural gene expression program has important implications for studies of neural development, neurological disease modeling and regenerative medicine.